NGF-induced mechanical sensitization of the masseter muscle is mediated through peripheral NMDA receptors

Highlights

• NGF induced long lasting mechanical sensitization in masseter muscle in rats.

• NGF-induced mechanical sensitization was greater in female than male rats.

• APV reversed NGF-induced mechanical sensitization in male but not female rats.

• NGF increased NMDA expression in masseter nociceptors in both sexes.

• NMDA expression in masseter nociceptors was similar between humans and rats.

Abstract

Intramuscular injection of nerve growth factor (NGF) in healthy humans mimics some of the symptoms of myofascial temporomandibular disorders (M-TMD). We hypothesized that NGF induces a prolonged myofascial mechanical sensitization by increasing peripheral N-methyl-d-aspartate (NMDA) receptor expression, leading to an enhanced response of muscle nociceptors to endogenous glutamate. Behavioral experiments with an injection of NGF (25 μg/ml, 10 μl) into the masseter muscle reduced the mechanical withdrawal threshold for 1 day in male rats and 5 days in female rats. These results mirror the sex-related differences found in NGF-induced mechanical sensitization in humans. Intramuscular injection with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV, 0.020 g/ml, 10 μl) reversed the mechanical sensitization in male but not in female rats. NGF increased the number of NMDA receptor subtype 2B (NR2B)-expressing rat trigeminal masseter ganglion neurons in both sexes, which peaked at 3 days post injection. There was an association between the levels of NR2B expression and NGF-induced mechanical sensitization. The average soma size of NR2B-expressing neurons increased significantly. Increased expression of neuropeptides (CGRP and SP) was observed in NR2B-expressing masseter ganglion neurons in female but not in male rats. In healthy men and women, comparable basal expression levels of NR2B and SP were found in peripheral fibers from masseter muscle microbiopsies. This study suggests that NGF-induced sensitization of masseter nociceptors is mediated, in part, by enhanced peripheral NMDA receptor expression. Measurement of peripheral NMDA receptor expression may be useful as a biomarker for M-TMD pain.

Introduction

Nerve growth factor (NGF) is a neurotrophin essential for the growth and survival of sympathetic and small diameter sensory afferent fibers (Bennett, 2001, Pezet and MacMahon, 2006). It may also play a role in chronic muscle pain development (Stohler, 1997). Recent clinical trials found anti-NGF antibodies to be efficacious in treating osteoarthritic pain and chronic low back pain (Lane et al., 2010, Katz et al., 2011). Systemic injection of NGF induced a long-lasting myalgia including jaw muscle pain (Petty et al., 1994). NGF injected into the masseter muscle of healthy subjects induced a local mechanical sensitization that lasted for 1–3 weeks as well as pain during yawning and chewing (Svensson et al., 2003, Svensson et al., 2008). These effects of NGF mimic some of the features of myofascial temporomandibular disorders (M-TMD), where patients suffer from a localized masseter muscle sensitivity upon palpation as well as pain during oral functions (Cairns, 2010). The magnitude of these symptoms was also greater in women than men (Svensson et al., 2003, Svensson et al., 2008). These results suggest that intramuscular NGF injections may be useful in modeling the tender points associated with M-TMD.

It is not clear how NGF injections induce prolonged mechanical sensitization of the masseter muscle. Injection of NGF into rat masseter muscle induced a TrkA receptor-mediated mechanical sensitization of nociceptors, which began within 30 min of injection and lasted for at least 3 h (Svensson et al., 2010). These findings suggest that a peripheral mechanism underlies the initiation of NGF-induced mechanical sensitization but do not shed light on its long duration. We speculate that the sensitizing effects of NGF might be due, in part, to an interaction between NGF and peripheral N-methyl-D-aspartate (NMDA) receptors on primary afferent nociceptors. Results from clinical and animal studies suggest that peripheral glutamate receptors might play a role in nociceptive signaling from musculoskeletal tissues in the orofacial region. NGF and glutamate are released during injury and inflammation and can be found in elevated levels in chronic pain conditions (Anand, 1995, Castrillon et al., 2010). Intramuscular injections of glutamate induced mechanical sensitization in the masseter muscle of humans and rats; however, the duration is only a few hours (Cairns et al., 2002, Cairns et al., 2003, Svensson et al., 2003). NGF-induced mechanical sensitization and glutamate-evoked discharge/pain both exhibited a sex-related difference with the greater response in females (Cairns et al., 2003, Svensson et al., 2003, Svensson et al., 2008). In rats, this difference appears to be due in part to an estrogen-mediated expression of NMDA receptor subtype 2B (NR2B) subunit containing NMDA receptors by masseter nociceptors in females (Woolley et al., 1997, Foy et al., 1999, Dong et al., 2007, McRoberts et al., 2007). Previous research has also found that the majority of peripheral NMDA receptors contain the NR2B subunit and that NMDA-evoked muscle afferent discharge can be attenuated by ifenprodil, a NR2B-selective NMDA antagonist, in both male and female rats (Li et al., 2004, Dong et al., 2007). In cultured neurons, NGF can induce release of glutamate through TrkA receptor activation (Numakawa et al., 2003, Raiteri et al., 2003) and can also increase NMDA receptor currents by phosphorylation and/or by increasing receptor expression (Bai and Kusiak, 1997, Jarvis et al., 1997, Di Luca et al., 2001). These results suggest that NGF could influence the excitability of masseter nociceptors by altering the activity of peripheral NR2B subunit containing NMDA receptors.

This study aimed to examine whether NGF induces a prolonged mechanical sensitization of rat masseter muscle by increasing NMDA receptor activity and if so, whether this sensitization exhibits a sex-related difference. The study further examined whether putative nociceptive fibers in the masseter muscle of healthy humans express NMDA receptors. Understanding the role of peripheral NMDA receptors in NGF-induced muscle pain may provide further insights into chronic muscle pain development.